The nucleus is the inner sanctuary of the eukaryotic cell, keeping the genetic information enclosed by the nuclear envelope (NE). The NE consists of two concentric membrane bilayers fused at specific sites to form circular openings occupied by the nuclear pore complex (NPC). As the only gateway into and out of the nucleus, the NPC plays a critical role in cellular homeostasis, but it is more than a transport organelle. We are beginning to appreciate that the NPC also serves as a reference point to organize the nucleus and is likely to play a pivotal role in gene regulation. This research program focuses on determining the molecular architecture of the enormous ~50 MDa NPC. Attaining this goal will provide a structural basis to interrogate its myriad functions in transport, gene regulation, and nuclear organization. Importantly, aberrant function of the NPC and its constituents is a primary cause of many human diseases, including leukemia, other cancers, autoimmune diseases, cardiomyopathies, and a variety of viral infections. The NPC is a modular structure, composed of ~30 different proteins, so-called nucleoporins that arrange in multiple copies around a central eightfold rotational axis. Subcomplexes of 2-10 nucleoporins hierarchically build up the NPC. Two of these subcomplexes, the heteromeric Y- and Nic96-complexes, organize the majority of architectural nucleoporins and establish the stable scaffold of the NPC. Consequently, these two subcomplexes are the main focus of this proposal. Once both are fully structurally characterized, the scaffold structure of the NPC can likely be generated with high confidence. This proposal is organized around four aims. Aim 1 - Structural characterization of the universally conserved, heptameric Y-complex core. The 575 kDa Y-complex is the pivotal NPC architectural scaffold unit. Building on recently published work on fragments of the Y-complex it is now feasible to structurally characterize the entire complex. A combined approach using crystallographic and electron microscopic methods will be employed. Aim 2 - Structural characterization of the species-specific Y-complex extensions. The human Y-complex contains three additional proteins, whose functional roles include NPC architecture and assembly. These proteins will be studied structurally in complex with the core components of the Y-complex. Aim 3 - Structural characterization of the Nic96 complex. The Nic96 complex is only partially characterized, but it shares common ancestry with the Y-complex. It is widely accepted that it is pivotal to elucidate the Nic96 complex structure as it is, beside the Y-complex, the second large building block of the NPC scaffold structure. Aim 4 - Assembly structure of the NPC and interactome. NPC subcomplexes have to interact to form the higher order assembly of the NPC, yet information on these inter-subcomplex contacts is still incomplete. To solve this problem, a yeast-two-hybrid based interactome will be generated sampling binary interactions between all nucleoporins.